Project Summary I. Training Program: My overall career goal is to uncover decisive regulators responsible for cancer progression, and seek for novel therapeutic targets for cancer treatment as part of the fight against breast cancer. Traditional breast cancer therapies successfully repress some tumors but often fail to eradicate this disease, possibly due to their inability to eliminate cancer stem cell populations. Therefore, novel therapeutic strategies aimed at targeting cancer stem cell may significantly increase cure and survival rate of cancer treatments. My mentors, Drs. Hui-Kuan Lin and Mien-Chie Hung are experienced in animal model of cancer and translational study, respectively. Moreover, both of them have demonstrated outstanding publication and training records in cancer oncology. In addition, there is a wealth of scientific support and technical assistance inside and outside the laboratory. Given the superb academic environment of M. D. Anderson and outstanding guidance from Drs. Lin and Hung provided to the PI, the PI has obtained well-rounded trainings essential for an independent researcher. The support of this research proposal will enable the PI to acquire further research independence and achieve full science maturation. II. Research Plan: Background: Epithelial to mesenchymal transition (EMT) is a critical process that accounts for breast cancer metastasis and progression, and recently is engaged in acquiring cancer stem cell (CSC) properties. CSCs recently have emerged as playing an important role in cancer initiation, progression, and relapse after chemotherapy. Thus, developing novel breast cancer therapies aimed at targeting CSC population will produce long-term tumor remission and increase survival rate of breast cancer patients. However, the regulatory machinery accounting for EMT-mediated CSC function is poorly understood. Although Twist is a main driver for EMT-mediated CSC generation whose upregulaiton associated with poor prognosis outcomes, how Twist activity is regulated, particularly at post-transcriptional level, remain unclear. This proposal is to address these outstanding questions. Hypothesis and Specific Aims: The central hypothesis of the application is that Skp2-promoted Twist polyubiquitination proceeds EMT reprogramming and acquires CSC properties, which consequently causes CSC-induce breast carcinogenesis. I plan to test our central hypothesis and accomplish the objective of this application by pursuing the following specific aims: Aim 1) To determine the functional link between Skp2 and Twist in EMT process and breast CSC functions. As Skp2 associates with and promotes Twist polyubiquitination; in this aim, I will determine whether critical role of Skp2 in promoting Twist ubiquitination and subsequent EMT process, and EMT-associated breast CSC functions. Aim 2) To delineate mechanism by which polyubiquitination regulates Twist-mediated EMT and cancer stem cell functions. K63-linked ubiquitination is known to modulate diverse cellular functions through regulating rafficking or signal activation. As Skp2 promotes K63-linked polyubiquitination of Twist and regulates CSC populations. In this aim, I will determine underlying mechanism of how K63-linked polyubiquitination regulates Twist function/activity. Additionally, I will identify the deubiquitinating enzymes that negatively regulate Twist functions through removal of its K63-linked ubiquitination. Aim 3) To assess in vivo roles of Twist polyubiquitination and Skp2 targeting in breast CSC functions and drug resistance. Recent advances showed that activation of EMT process and increase of CSC populations cause drug resistance in various cancer types. In this aim, I will further examine the role of Twist polyubiquitination in CSC regulation in vivo. Additionally, I will examine whether Skp2 targeting eradicates breast CSC pools and re-sensitizes breast tumor responses to current treatments. Impact: This project covers thorough trainings from basic molecular mechanisms to preclinical animal studies, which fully prepare me toward the fight against breast cancer. This proposal project is innovative, because it deciphers novel regulatory modes of breast cancer stem cells, a newly established field in breast cancer research. The success of this proposal not only will conceptually advance current understanding toward regulations of CSCs, but also could be applied to overcome drug resistance and later tumor relapse in breast cancer patients.